The present invention involves processes to detect nucleic acid sequences and antibodies in samples by amplifying signals associated with the existence of such nucleic acid sequences and antibodies. In particular, an oligonucleotide sequence is bound to the nucleic acid sequence to be detected or, through a connector molecule, to an antibody to be detected, and that oligonucleotide is used to form a double-stranded nucleic acid sequence which is used to synthesize relatively large quantities of RNA transcripts in a short period of time for detection.
Many target and signal amplification techniques have been described in the literature, but none of these techniques is believed to offer the combination of specificity, simplicity, and speed of the present invention. Some of these various techniques are described below.
a) Polymerase Chain Reaction (PCR) PCR is described in Saiki et al. (1985), Science, 230 1350. PCR consists of repeated cycles of DNA polymerase generated primer extension reactions. The target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridized. These oligonucleotides become primers for use with DNA polymerase. The DNA is copied by primer extension to make a second copy of both strands. By repeating the cycle of heat denaturation, primer hybridization and extension, the target DNA can be amplified a million fold or more in about two to four hours. PCR is a molecular biology tool which must be used in conjunction with a detection technique to determine the results of amplification. The advantage of PCR is that it may increase sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in about 4 hours. The disadvantage is that contamination may result in false positive results (i.e., reduced specificity).
b) Transcription Amplification (TAS) TAS utilizes RNA transcription to amplify a DNA or RNA target and is described in Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173. TAS uses two phases of amplification. In phase 1 of TAS a duplex cDNA is formed containing an overhanging, single stranded T7 transcription promoter by hybridizing a polynucleotide to the target. The DNA is copied by reverse transcriptase into a duplex form. This is heat denatured and a primer for the opposite strand from that with the T7 region is hybridized. Using this primer, reverse transcriptase is again added to create a double stranded cDNA, which now has a double stranded (active) T7 polymerase binding site. T7 RNA polymerase transcribes the duplex to create a large quantity of single stranded RNA. This is the completion of phase one of TAS. In phase 2, the primer is again used. This time it is hybridized to the new RNA and again converted to duplex cDNA. The duplex is heat denatured and the cycle is continued as before. In contrast to PCR where two copies of the target are generated each cycle, the advantage of TAS is that between 10 and 100 copies of each target molecule are produced with each cycle. This means that 10.sup.6 fold amplification can be achieved in only 4 to 6 cycles, but this still takes 3-4 hours. The major disadvantage of TAS is the number of enzyme addition steps and the heat denaturation requirements.
c) Transcriptions Amplification (3SR) In a modification of TAS, known as 3SR, enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation, as described in Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874. RNAse H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplification of 10.sup.6 to 10.sup.9 have been achieved in 1 hour at 42.degree. C.
d) Ligation Amplification (LAR/LAS) Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described in Wu, D. Y. and Wallace, R. B. (1989) Genomics 4, 560. The oligonucleotides hybridize to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated. LAR suffers from the fact that the ligases can join the oligonucleotides even when they are not hybridized to the target DNA. This results in a high background. In addition, LAR is not an efficient reaction and therefore currently requires about five hours for each cycle. Thus, the amplification takes a couple of days.
e) Q Beta RNA Replication In this technique, RNA replicase for the bacteriophage Q Beta, which replicates single stranded RNA, is used to amplify the target DNA, as described in Lizardi et al. (1988) Bio/Technology 6, 1197. First, the target DNA is hybridized to a primer containing T7 promoter and a Q Beta 5' sequence region. Using this primer, reverse transcriptase generates a cDNA connecting the primer to its 5' end in the process. These two steps are similar to the TAS protocol. The resulting heteroduplex is heat denatured. Next, a second primer containing a Q Beta 3' sequence region is used to initiate a second round of cDNA synthesis. This results in a double stranded DNA containing both 5' and 3' ends of the Q-Beta bacteriophage as well as an active T7 RNA polymerase binding site. T7 RNA polymerase then transcribes the ds-DNA into new RNA, which mimics the Q Beta virus. After extensive washing to remove any unhybridized probe, the new RNA is eluted from the target and replicated by Q Beta replicase. The latter reaction created 10.sup.7 amplification in 20 minutes. Significant background may be formed due to minute amounts of probe RNA that is non-specifically retained during the reaction.
f) Chiron Signal Amplification The Chiron system, as described in Urdea et al. (1987) Gene 61, 253, is extremely complex. It utilizes 12 capture oligonucleotide probes, 36 labeled oligonucleotides, 20 biotinylated immobilization probes that are cross-linked to 20 more enzyme labeled probes. This massive conglomerate is built-up in a stepwise fashion requiring numerous washing and reagent addition steps. Amplification is limited because there is no cycle. The probes simply form a large network.
g) ImClone Signal Amplification ImClone utilizes a network concept similar to Chiron, but the approach is completely different. The ImClone technique is described in Kohlbert et al. (1989) Mol and Cell Probes 3, 59. ImClone first binds a single stranded M13 phage DNA containing targeted probe. To this bound circular DNA is then hybridized about five additional DNA fragments that only bind to one end and the other end hangs freely out in the solution. Another probe set is then hybridized to the hanging portion of the previous set of probes. The latter set is either labeled directly with an enzyme or it is biotinylated. If it is biotinylated, then detection is via a streptavidin enzyme complex. In either case, detection is through an enzyme color reaction. Like the Chiron method, ImClone relies on build-up of a large network. Because there is no repeated cycle, the reaction is not geometrically expanded, resulting in limited amplification.